Human CXCR4 Stable Cell Line-HEK293

C-X-C chemokine receptor type 4 (CXCR4) is an emerging target for anticancer drug development due to its high expression in cancer cells. Here, researchers aimed to generate CXCR4 overexpressing HEK293T cells for use in nonradioactive binding assays, building a platform for identifying CXCR4-targeting drug candidates. The resulting CXCR4-overexpressing HEK293T cells demonstrated high expression (99.8%). The IC50 value of plerixafor was determined using a fluorescently labeled antibody competition assay using CXCR4-overexpressing HEK293T cells and is consistent with previously reported values using radioligand binding assays. No significant displacement of bound PE-anti-hCD184 was observed with the tested native compounds, potentially due to nonspecific binding to other functional targets or organelles, lower potency, or binding to CXCR4 in a deeper pocket. Therefore, a validated nonradioactive binding assay can serve as an alternative screening tool for anticancer lead compounds targeting CXCR4 and is also an important tool for proof of mechanism studies in drug discovery.

Although the natural compounds tested in this study exhibited low binding affinity for CXCR4, they may possess advantages in terms of selectivity for cancer cells over normal cells. Therefore, the researchers further evaluated the binding properties of the test natural compounds to strengthen their potential for CXCR4 inhibition. Significant differences in the fluorescence intensity signal of PE-anti-hCD184 between normal and CXCR4-overexpressing HEK293T cells were observed, indicating that the antibody possesses high selectivity for CXCR4 (Figure 1A). This suggests that the developed CXCR4-overexpressing HEK293T cells are relevant for further evaluation of compounds with potential CXCR4 inhibitory activity. CXCR4-overexpressing HEK293T cells treated with the test compounds demonstrated reduced affinity for PE-anti-hCD184 compared to normal HEK293T cells treated with the same compounds. The lower displacement of the PE-conjugated antibody observed in competition binding assays with the test compounds may indicate weaker CXCR4 binding affinity. These results also indicate that the developed CXCR4-overexpressing HEK293T cells can be used as a non-radioactive ligand binding assay platform for screening novel CXCR4 ligands.

Figure 1. Competition binding assay of PE-anti-hCD184 and the natural compounds to CXCR4. (Ha D T T, et al., 2022)